The long term objective of this research is to understand the mechanism and control of intracellular apolar lipid transport and the means by which lipids and proteins are assembled into nascent plasma lipoproteins in the liver and small intestine. Lipoprotein metabolism will not be fully understood until the pathways of assembly, the various factors involved, and the regulation of assembly are delineated. The mechanism of plasma lipoprotein assembly is currently unknown. A microsomal lipid transfer protein (MTP) which transports triglyceride (TG) and cholesteryl ester (CE) between synthetic membranes (1,2) has been identified, isolated from bovine liver, and characterized. The properties of MTP suggest that it may play a role in the assembly of the triglyceride-rich lipoproteins, chylomicrons and very low density lipoprotein (VLDL). This application proposes to continue the investigation of MTP by further defining the structure and activity of human MTP. An understanding of the structure and function of this protein should provide new insights into its role in the assembly of triglyceride- rich lipoproteins. Initially, structural or functional relationships between the two subunits of MTP will be identified. Using peptide mapping and protein sequencing techniques, it will be determined if the two subunits are structurally unique or if one subunit is homologous to the other subunit or a plasma lipid transfer protein. Using polyclonal antibodies specific for each subunit and isolated MTP subunits, the subunits which possesses the TG and CE transfer activities will be determined. Kinetic studies will be used to define the mechanism by which TG and CE are transferred between membranes. By testing the effect of substrates of varied compositions on lipid transfer, an understanding of the effect of the membrane structure on the rate of apolar lipid transfer will be developed. These studies will extend previous studies designed to determine if the transfer properties of MTP are consistent with a protein involved in lipoprotein biogenesis.